Borrelia burgdorferi (Bb), the Lyme disease (LD) spirochete, is maintained in nature via an enzootic cycle which typically involves wild rodents and Ixodes ticks. During the current funding interval, we have used an expanding armamentarium of model systems and genetic tools to define the physiological and virulence-related functions of Bb genes involved in mammalian host adaptation as well as the mechanisms that underlie the spirochete's transition from arthropod vector to mammalian host. Much of this work has centered about delineating the relative contributions of the RpoS-dependent and -independent transcriptional pathways to the processes which enable spirochetes to transit from tick to mammal. Microarray-based transcriptional profiling of wild-type and rpoS mutant spirochetes grown in dialysis membrane chambers (DMCs) has enabled us to define the in vivo RpoS regulon, the cohort of genes that the alternate sigma factor RpoS controls, both positively and negatively, in response to mammalian host-derived signals. By extrapolating from these and related findings to the scenario of the infected nymphal tick, we have formulated our central hypothesis: beginning with the taking of the blood meal, RpoS acts as a "gatekeeper" that coordinates the reciprocal upregulation and repression of a subset of differentially expressed borrelial genes required to establish mammalian infection. As a corollary to this hypothesis, we propose that the "RpoS off'state represents an alternate developmental program required by the spirochete to establish the tick-phase of the enzootic life cycle. The principal long-term objective of our Research Plan is to integrate the search for novel Bb virulence determinants that function within the tick or the mouse with efforts to decipher the mechanisms that underlie differential gene expression by the LD spirochete. To accomplish this objective, we have formulated three Specific Aims: (i) to further characterize genes within the in vivo RpoS regulon which we hypothesize are induced by the blood meal and promote infection of the mammalian host;(ii) to further characterize genes which require RpoS for repression in vivo and which we hypothesize are required for the tick- phase of the spirochete's life cycle;and (iii) to develop a broader understanding of the RpoS regulon by delineating its "on" and "off" states during the enzootic cycle and examining selected facets of RpoS function.